Enhanced ZNF521 expression induces an aggressive phenotype in human ovarian carcinoma cell lines

Epithelial ovarian carcinoma (EOC) is the most lethal gynecological tumor, that almost inevitably relapses and develops chemo-resistance. A better understanding of molecular events underlying the biological behavior of this tumor, as well as identification of new biomarkers and therapeutic targets are the prerequisite to improve its clinical management. ZNF521 gene amplifications are present in >6% of OCs and its overexpression is associated with poor prognosis, suggesting that it may play an important role in OC. Increased ZNF521 expression resulted in an enhancement of OC HeyA8 and ES-2 cell growth and motility. Analysis of RNA isolated from transduced cells by RNA-Seq and qRT-PCR revealed that several genes involved in growth, proliferation, migration and tumor invasiveness are differentially expressed following increased ZNF521 expression. The data illustrate a novel biological role of ZNF521 in OC that, thanks to the early and easy detection by RNA-Seq, can be used as biomarker for identification and treatment of OC patients.


Introduction
Epithelial ovarian carcinoma is the leading cause of gynecological cancer-related mortality, with 295,000 new cases and 184,000 deaths worldwide in 2020 [1]. Due to the lack of effective screening programs two-thirds of patients with OC are diagnosed with advanced disease. Despite a generally good response to surgery and first-line chemotherapy, OC almost inevitably relapses in different peritoneal sites and develops chemo-resistance. Only 45% of OCaffected women are likely to survive for five years, compared to 89% of breast cancer patients [2,3]. Despite increased radicality of debulking surgery and modifications of conventional chemotherapies there have been only modest gains for overall survival (OS) in OC in the last decades [4]. Based on their histological and morphological classification OC can be divided in serous, mucinous, endometrial, clear cells and transitional cells (depending on the tissue of origin involved). Moreover, OCs can be divided in type I (low-grade tumor with BRAF and KRAS mutations) or type II (high-grade tumor with a variety of mutations including HOX, PTEN, KRAS, AKT1, BRCA1/2 genes) [5][6][7][8][9][10] and various dysregulated mechanisms underlying the pathology [11][12][13]. An important therapeutic target in OC may be represented by a subpopulation of cells defined as ovarian cancer stem cells (OCSCs) [14] and considered a key factor in cancer initiation, relapse and resistance to therapy [15,16]. The stem cell-associated transcription co-factor, zinc finger protein 521 (ZNF521) is a transcription factor abundant in OC, where a high expression is associated with poor prognosis [17,18] Additionally, in ovarian cancer an integration analysis of microRNA and mRNA gave a positive association of ZNF521 with the miR34-family and miR133b [18].
As the mortality rate of this tumor has improved only marginally over the past decades, a better understanding of the molecular events that drive its development, maintenance and progression, and the identification of new biomarkers and therapeutic targets, are paramount to improve OC treatment and hence its prognosis. In this study the role of ZNF521 is investigated in the regulation of human ovarian carcinoma cells.

Cell lines and culture conditions
The differentiated papillary human ovary cystoadenocarcinoma cell line HeyA8, the human desmoplastic cerebellar medulloblastoma DAOY cells and the human embryonic kidney HEK293T cells were cultured in DMEM. The human ovarian adenocarcinoma cell line ES-2, the acute monocytic leukemia THP1 cells and the lymphoblastoid B (multiple myeloma) IM9 cells were cultured in RPMI. Cell culture medium are supplemented with 10% fetal bovine serum, 50U of penicillin and 50μg of streptomycin/ml (Thermo Fisher Scientific, Milan, Italy) and cell lines were maintained at 37˚C in 5% CO 2 .

Transfection and transduction of cell lines
Lentiviral production was carried out in 100 mm tissue culture plates, where the cell line HEK293T were transfected with 10μg of plasmid for FUIGW (control vector) or FUIGW-ZNF521 [40] plus lentiviral packaging plasmids (2μg of pCMV-VSVG and 10μg of pCMV-deltaR8-91), using the calcium phosphate method. Lentiviral media were collected at 24, 48 and 72 hours after transfection and supplemented with 6μg/ml of polybrene [41] and ovarian carcinoma cell lines HeyA8 and ES-2 were stably transduced with the control vector FUIGW or FUIGW-ZNF521. Three rounds of transduction were performed by centrifuging the cells with lentivirus at 3200 rpm at 32˚C for 50min. Cells were 70-80% positive for the transgene EGFP by FACS analysis and stably expressed the ZNF521 protein. The transduction was performed in three independent experiments and cells were further sorted for EGFP giving a homogeneous population over 90% positive.

Proteins extracts and Western blotting
HeyA8 and ES-2 cells were processed for nuclear and cytoplasmic extracts. Cells were scraped, resuspended in hypotonic lysis buffer 10mM HEPES pH7.9, 10mM KCl, 0.1mM EDTA, protease inhibitors (P8849, Sigma-Aldrich) and phosphatase inhibitor cocktails 2 and 3 (P0044, P5726, Sigma-Aldrich) and incubated on ice for 20 min. After the addition of 0.25% Igepal-630 (NP40) (Sigma-Aldrich), samples were centrifuged at 3000 rpm for 5 min and supernatants containing the cytoplasmic extracts were recovered. Nuclear pellets were resuspended in 20mM HEPES pH7.9, 0.4M NaCl, 1mM EDTA with protease and phosphatase inhibitors. After three cycles of vortex and ice, samples were centrifuged at 12000 rpm for 20 min and the supernatants containing the nuclear extracts were collected.

Cell proliferation assay
Transduced cells (HeyA8 and ES-2) were seeded into 6 well plates at 2.0x10 5 cells/well in complete DMEM or RPMI respectively. Every 48 hours cells were trypsinized, counted and replated at 2.0x10 5 /well. The average of three independent cell counts, normalized to account for the dilutions performed in the individual cultures at each replating, was plotted against time to obtain a cumulative growth curve for each cell population.
The growth of HeyA8 was also evaluated using the Real-Time Glo MT Cell Viability Assay (Promega, Milan, Italy) kit, which allows the evaluation of cell proliferation based on their ATP consumption. The cells were plated in 96 wells and then the substrate and the enzyme are added in a 1:1 ratio. The plate was then incubated at 37˚C and the readings (GloMax Explorer, Promega) taken at 0, 24 and 48 hours of growth.

Wound healing assay-migration assay
Transduced cells were counted, seeded at the same density and cultured until >90% confluence in 6 well plates in appropriate medium with 10% FBS and then in DMEM or RPMI with reduced serum 0.5% FBS to block their proliferation. After 24 hours the monolayer of cells was scratched with a sterile 200μl pipet tip, washed with PBS 1X and incubated at 37˚C in DMEM 10% FBS [43] Images were captured by phase contrast microscopy at 10x magnification, at the beginning and at regular intervals during cell migration. The migration of the cells was analysed and quantified by ImageJ 1.51j8. Experiments were performed in triplicate.

Next generation sequencing-total RNA-Seq
RNA concentration was determined by using Quant-IT RNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies) and its quality and integrity assessed with the Agilent 4200 Tapestation System (Agilent Technologies). Total RNA-seq procedures were performed as described previously [44]. Indexed libraries were prepared using 1 μg of total RNA in technical and biological triplicate for each sample as starting material, with a TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc.). Libraries were sequenced (paired-end, 2x75 cycles) at a concentration of 8 pM/lane on NextSeq 500 platform (Illumina Inc.) [45]. Aliquots of the amplified RNA samples used for RNA-Seq remained available to be used to confirm by qRT-PCR the differential expression of selected genes.
Fastq files were aligned to the hg38 genome assembly using STAR [46]. STAR gene counts were normalized applying the median of ratios method implemented in DESeq2 R package [47]. Briefly, the normalization process implies different steps: i) for each gene, a pseudo-reference sample is created and is equal to the geometric mean across all samples; ii) for every gene in a sample and for each sample, the ratios sample/ref are calculated; iii) the median value of all ratios for a given sample is taken as the normalization factor (size factor) for that sample; iv) for each gene in each sample the normalized count values is calculated dividing each raw count value by the sample's normalization factor.
For differential gene expression (DEG) analysis, we applied DESeq2 R package to normalized counts.
Differentially expressed genes were used as input for the Molecular Signature Database Investigate gene sets analysis [48].

Expression analysis by qRT-PCR
cDNA was synthesized using SuperScript III reverse transcriptase at 42˚C and 2.5μM random hexamers (Thermo Fisher Scientific) from 1μg RNA previously prepared with Tri Reagent (Sigma-Aldrich) and verified by the NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific) [49]. One cycle of 3 min at 95˚C was followed by 45 cycles of 10 sec at 95˚C, 10 sec at 60˚C and 10 sec at 72˚C, finishing with a melting curve, amplified with the iQ™ SYBR1 green super mix (BioRad, Milan, Italy) using the qRT-PCR amplifier QuantStudio3 (Applied Biosystems, Milan, Italy).
Analysis of gene expression was calculated as 2 −ddCt and normalized for the housekeeping gene (GAPDH). Primers used in this study were as follows (5'-3'): h-ZNF521 were previously described [50]; Experiments were performed in triplicate.

Enforced expression of ZNF521 enhances growth of ovarian carcinoma cell lines
ZNF521 is expressed in ovary and ovarian cancers [18] and mutations of its gene (almost invariably amplifications) are present in >6% of OCs (S1A Fig) [20]. Increased expression of ZNF521 was found associated with a poor prognosis in OC patients [17][18][19]. Analysis of a public genomic database [51] comprising 655 OC patients confirmed an adverse prognosis for patients with ZNF521-high OCs (S1B Fig) [52].
To assess whether the modulation of ZNF521 expression could affect some biological characteristics of OC cells the mRNA levels of ZNF521 for HeyA8 and ES-2 OC cells were quantified by qRT-PCR and compared to that of THP1 cells, DAOY cells and of IM9 that, respectively, express very high, intermediate and very low levels of ZNF521 (Fig 1A). The HeyA8 and ES-2 cell lines, were stably transduced with two different lentiviral vectors: FUIGW (carrying only the EGFP) or FUIGW-ZNF521 (expressing the ZNF521 cDNA fused to 3xFLAG tag and EGFP) and immunoblot analysis as well as qRT-PCR confirmed a significant increase of ZNF521 expression (Fig 1B and 1C).

ZNF521 improves the migration ability of human ovarian cell lines
The cell migration and invasion of ZNF521-overexpressing and CTL cells was investigated by wound-healing assay. Cells were plated and grown to confluence, in 6 well plates, and then starved for 24 hours. When cell confluence approximately reached 90%, a longitudinal wound was done using a sterile tip. Cells cultures were observed at regular time intervals, monitoring the extent of the healing of the wound. Both ZNF521-overexpressing cell lines displayed ã 2-fold increase in wound healing capacity (Fig 3) compared to CTL cells.

ZNF521 enhances clonogenicity of HeyA8 and ES-2 cells
The proliferation of HeyA8 and ES-2, ZNF521-overexpressing or CTL cells was assessed in anchorage-independent conditions to test their ability to form spheroids as readout for enrichment in CSC subpopulation in OC cell lines. . "T" in 1T, 2T or 3T indicate each "Transduction". The two bands for ZNF521 correspond to the monomer and dimers at 150kDa and 300kDa identified by the S15 anti-ZNF521 antibody. Overall, ZNF521-overexpressing cells showed a greater number of spheroids and of cells forming spheres (Fig 4A, 4B, 4D and 4E) compared to the controls which confirmed an  Notably, ZNF521 transduced cell lines formed larger spheres than the controls (Fig 4C and  4F).

ZNF521 over-expression reprograms the transcriptome of HeyA8 and ES-2 cells
Since ZNF521 is a transcription co-factor we then sought at the transcriptome of OC cell lines upon ZNF521 expression modulation. RNA-Seq analysis of both ES-2 and HeyA8 cell lines, ZNF521-OE or CTL, revealed that ZNF521 overexpression induced a large transcriptional reprogramming involving a total of 1375 and 742 differentially expressed genes (p<0.05), respectively ( Fig 5A). Furthermore, Molecular Signature Database (MSigDB) focused on hallmark gene sets (see Methods) scored the enrichment of 4 hallmarks related to cancer proliferation (i.e., E2F targets, EMT, G2M checkpoint and mitotic spindle) both in HeyA8 and in ES-2 cell lines (Fig 5B and 5C), which is in line with previously described enhanced proliferation and migration of HeyA8 and ES-2 cells upon ZNF521 overexpression (Figs 2 and 3). Yet, a total of 106 genes were found overlapping between the 1375 and 742 differentially expressed genes in ZNF521-OE cells (p<0.0001, Fisher's exact test; Fig 5D). Of these, 10 genes previously described to be associated to tumour growth, metastasis, cell migration and proliferation, were found consistently down-or up-modulated in ZNF521-OE cells vs CTLs (Fig 5E). Lastly,

Discussion
Ovarian carcinoma represents an important cancer-related cause of death in women, even considering that the overall survival has only modest advantage from radical debulking surgery and conventional chemotherapy [4]. The major unresolved clinical problems include malignant progression and rapid emergence of drug resistance against conventional chemotherapy. In this regard, novel strategies are warranted based on molecularly targeted therapies directed against components of specific signaling pathways required for tumour development and progression [53]. Additionally, identification of biomarkers with functional and prognostic significance, may greatly aid early diagnosis and allow timely implementation of therapeutic strategies. Profiling of 157 advanced stage serous ovarian cancers, ZNF521 (known regulator of the homeostasis in stem cells compartment and in cancer [18,22-28]) results in a set of 86-genes with significant poor overall survival profiles [17]. In exosomes derived from Bone Marrow Stromal Cells (BMSCs), ZNF521 was identified as hub protein and associated with poor outcome in gastric and ovarian cancers [18] where OCSC are shown to be implicated in the progression of ovarian carcinoma and resistance to therapy [14-16]. Moreover, recently, ZNF521 was identified as one of the top 15 genes significantly correlated with the overall survival in 1692 serous ovarian cystadenocarcinomas [19]. Taken together the analyses of different public genomic databases indicate that ZNF521 is: i) amplified in 6% of ovarian carcinoma cases and ii) associated to poor survival in ovarian cancer patients [18-20] (S1 Fig). Here, we highlight a new role of the transcriptional co-factor ZNF521 in OC. Overall, we found that higher levels of ZNF521 in HeyA8 and ES-2 human ovarian cancer cell lines enhances their growth (Fig 2), ability to migrate (Fig 3) and to form OC spheroids (Fig 4). Such a role of ZNF521 in hOC may be exerted via the modulated expression of several regulatory genes which are relevant in cancer [54,55] (Fig 5 and S1-S3 Tables).
qRT-PCR analysis confirmed the regulation by ZNF521 of a subset of these genes by ZNF521 (N = 7; Fig 6) that, once dysregulated, induce cellular transformation and tumorigenesis.
Human OC HeyA8 and ES-2 cell lines overexpressing ZNF521 also increase their ability to form spheroids (enrichment of CSCs). CSCs have the ability to self-renew, differentiate and possess tumorigenic properties. CSCs are also involved in drug resistance, and it has been shown that some populations of CSCs share EMT-like cellular characteristics [82]. Indeed, whole-transcriptome profiling of OC cell lines confirmed the profound transcriptional impact of ZNF521 expression modulation on a set of genes involved in tumour growth, proliferation, HeyA8 (blu circle) and 742 genes were differentially expressed in ES-2 (red circle); 106 genes were differentially expressed in common in both cell lines. (E) Heatmap of DEGs specifically involved in growth, metastasis, migration and proliferation commonly regulated in both OC cell lines. FC, fold change (ZNF521 vs control).
https://doi.org/10.1371/journal.pone.0274785.g005 migration and metastasis. Forced ZNF521 expression altered the transcriptional profile of a total of 1375 and 742 genes in HeyA8 and in ES-2 human OC cell lines, respectively, as well as the enrichment of several cancer-related hallmarks including EMT regulatory genes.
Further in-depth analysis is necessary to better understand the molecular mechanism that underlines these activities of ZNF521 in human OC.
Taken together, these data are the first evidence of new role of the stem cell transcription co-factor ZNF521 (so far known for its involvement in the stem pathway) in the regulation of human epithelial ovarian carcinoma (hEOC) cells, suggesting that this could be due to the modulation exerted by ZNF521 on regulatory genes of the relevant molecular mechanisms of EMT in human ovarian cancer.